The present invention relates to a new complex receptor polypeptide LSR (Lipolysis Stimulated Receptor), characterized by its functional activities, the cloning of the cDNAs complementary to the messenger RNAs encoding each of the subunits of the multimeric complex, vectors and transformed cells, methods of diagnosis and of selection of compounds which can be used as medicament for the prevention and/or treatment of pathologies and/or of pathogeneses such as obesity and anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, and more generally the various pathologies associated with abnormalities in the metabolism of cytokines.
Obesity is a public health problem which is both serious and widespread: in industrialized countries, a third of the population has an excess weight of at least 20% relative to the ideal weight. The phenomenon continues to worsen, in regions of the globe whose economies are being modernized, such as the Pacific islands, and in general. In the United States, the number of obese people has passed from 25% at the end of the 70s to 33% at the beginning of the 90s.
Obesity considerably increases the risk of developing cardiovascular or metabolic diseases. It is estimated that if the entire population had an ideal weight, the risk of coronary insufficiency would decrease by 25% and that of cardiac insufficiency and of cerebral vascular accidents by 35%. Coronary insufficiency, atheromatous disease and cardiac insufficiency are at the forefront of the cardiovascular complications induced by obesity. For an excess weight greater than 30%, the incidence of coronary diseases is doubled in subjects under 50 years. Studies carried out for other diseases are equally eloquent. For an excess weight of 20%, the risk of high blood pressure is doubled. For an excess weight of 30%, the risk of developing a non-insulin-dependent diabetes is tripled. That of hyperlipidemias is multiplied by 6.
The list of diseases whose onset is promoted by obesity is long: hyperuricemia (11.4% in obese subjects, against 3.4% in the general population), digestive pathologies, abnormalities in hepatic functions, and even certain cancers.
Whether the physiological changes in obesity are characterized by an increase in the number of adipose cells, or by an increase in the quantity of triglycerides stored in each adipose cell, or by both, this excess weight results mainly from an imbalance between the quantities of calories consumed and those of the calories used by the body. Studies on the causes of this imbalance have been in several directions. Some have focused on studying the mechanism of absorption of foods, and therefore the molecules which control food intake and the feeling of satiety. Other studies have been related to the basal metabolism, that is to say the manner in which the body uses the calories consumed.
The treatments for obesity which have been proposed are of four types. Food restriction is the most frequently used. The obese individuals are advised to change their dietary habits so as to consume fewer calories. This type of treatment is effective in the short-term. However, the recidivation rate is very high. The increase in calorie use through physical exercise is also proposed. This treatment is ineffective when applied alone, but it improves, however, weight loss in subjects on a low-calorie diet. Gastrointestinal surgery, which reduces the absorption of the calories ingested, is effective but has been virtually abandoned because of the side effects which it causes. The medicinal approach uses either the anorexigenic action of molecules involved at the level of the central nervous system, or the effect of molecules which increase energy use by increasing the production of heat. The prototypes of this type of molecule are the thyroid hormones which uncouple oxidative phosphorylations of the mitochondrial respiratory chain. The side effects and the toxicity of this type of treatment make their use dangerous. An approach which aims to reduce the absorption of dietary lipids by sequestering them in the lumen of the digestive tube is also in place. However, it induces physiological imbalances which are difficult to tolerate: deficiency in the absorption of fat-soluble vitamins, flatulence and steatorrhoea. Whatever the envisaged therapeutic approach, the treatments of obesity are all characterized by an extremely high recidivation rate.
The molecular mechanisms responsible for obesity in humans are complex and involve genetic and environmental factors. Because of the low efficiency of the treatments known up until now, it is urgent to define the genetic mechanisms which determine obesity, so as to be able to develop better targeted medicaments.
More than 20 genes have been studied as possible candidates, either because they have been implicated in diseases of which obesity is one of the clinical manifestations, or because they are homologues of genes involved in obesity in animal models. Situated in the 7q31 chromosomal region, the OB gene is one of the most widely studied. Its product, leptin, is involved in the mechanisms of satiety. Leptin is a plasma protein of 16 kDa produced by the adipocytes under the action of various stimuli. Obese mice of the ob/ob type exhibit a deficiency in the leptin gene; this protein is undetectable in the plasma of these animals. The administration of leptin obtained by genetic engineering to ob/ob mice corrects their relative hyperphagia and allows normalization of their weight. This anorexigenic effect of leptin calls into play a receptor of the central nervous system: the ob receptor which belongs to the family of class 1 cytokine receptors. The ob receptor is deficient in obese mice of the db/db strain. The administration of leptin to these mice has no effect on their food intake and does not allow substantial reduction in their weight. The mechanisms by which the ob receptors transmit the signal for satiety are not precisely known. It is possible that neuropeptide Y is involved in this signalling pathway. It is important to specify at this stage that the ob receptors are not the only regulators of appetite. The Melanocortin 4 receptor is also involved since mice made deficient in this receptor are obese (Gura, 1997).
The discovery of leptin and the characterization of the leptin receptor at the level of the central nervous system have opened a new route for the search for medicaments against obesity. This model, however, rapidly proved disappointing. Indeed, with only one exception (Montague et al., 1997), the genes encoding leptin or its ob receptor have proved to be normal in obese human subjects. Furthermore and paradoxically, the plasma concentrations of leptin, the satiety hormone, are abnormally high in most obese human subjects. Most of the therapeutic research efforts in this direction have centred on the characterization of the effect of leptin at the level of the central nervous system.
The present invention results from a focusing of the research effort on the discovery of the mechanisms of leptin elimination. The most widely accepted working hypothesis is that the plasma levels of leptin are high in obese subjects because this hormone is produced by the adipose tissue and that the fatty mass is increased in obese subjects. The inventors have formulated a different hypothesis and have postulated that the concentrations of leptin are increased in obese individuals because the clearance of this hormone is reduced. This deficiency causes a leptin resistance syndrome and the obese individual develops a suitable response to the high concentrations of leptin. In this perspective, the treatment of obese subjects ought to consist not in an increase in the leptin levels but in a normalization thereof. At this stage, it is essential to recall that the ob type receptors are signalling type receptors. These receptors can bind leptin at the level of the plasma membrane but cannot cause the protein to enter inside the cell for it to be degraded therein. The ob receptors are not endocytosis receptors.
The inventors have characterized a receptor, in particular hepatic, called LSR receptor, whose activity is dual. The LSR receptor allows, on the one hand, endocytosis of lipoproteins, when it is activated by the free fatty acids, thus serving as a pathway for the clearance of lipoproteins. This pathway serves mainly, but not exclusively, for the clearance of particles high in triglycerides of intestinal origin (Mann et al., 1995). This activity, expressed most particularly at the hepatic level, is dependent on the presence of free fatty acids which, by binding to the receptor, induce a reversible change in the conformation of this complex and allow it to bind, with a high affinity, various classes of lipoproteins such as those containing apoprotein B or apoprotein E.
On the other hand, under normal conditions, in the absence of free fatty acids, the complex receptor LSR does not bind lipoproteins, but is capable of binding a cytokine, in particular leptin, and then of internalizing it and of degrading it.
The present invention therefore relates to a purified LSR receptor, in particular of hepatic cells, characterized in that it is capable, in the presence of free fatty acids, of binding lipoproteins, and in the absence of free fatty acids, of binding a cytokine, preferably leptin.
According to the invention, this LSR receptor is, in addition, characterized in that the bound lipoproteins or the bound cytokine are incorporated into the cell and then degraded, the bound lipoproteins containing in particular apoprotein B or E.
It should be understood that the invention does not relate to the LSR receptors in a natural form, that is to say that they are not taken in their natural environment but obtained by purification from natural sources, or alternatively obtained by genetic recombination, or alternatively by chemical synthesis and capable, in this case, of containing non-natural amino acids, as will be described below. The production of a recombinant LSR receptor, which may be carried out using one of the nucleotide sequences according to the invention, is particularly advantageous because it makes it possible to obtain an increased level of purity of the receptor.
More particularly, the invention relates to a purified rat LSR receptor, characterized in that it comprises at least one subunit having a molecular weight of about 66 kDa and a subunit having a molecular weight of about 58 kDa.
Preferably, the purified rat LSR receptor of the present invention is characterized in that it contains an xcex1 subunit comprising the amino acid sequence of SEQ ID 2 or a sequence homologous thereto, or an xcex1xe2x80x2 subunit comprising the amino acid sequence of SEQ ID 4 or a sequence homologous thereto, and one, preferably three, subunits comprising the amino acid sequence of SEQ ID 6 or a sequence homologous thereto.
The invention also relates to a purified mouse LSR receptor, characterized in that it comprises at least one subunit having a molecular weight of about 66 kDa and a subunit having a molecular weight of about 58 kDa.
Preferably, the purified mouse LSR receptor of the present invention is characterized in that it contains an xcex1 subunit comprising the amino acid sequence of SEQ ID 16 or a sequence homologous thereto, or an xcex1 subunit comprising the amino acid sequence of SEQ ID 17 or a sequence homologous thereto, and one, preferably three, xcex2 subunits comprising the amino acid sequence of SEQ ID 18 or a sequence homologous thereto.
The invention also relates to a purified human LSR receptor, characterized in that it comprises at least one subunit having a molecular weight of about 72 kDa and a subunit having a molecular weight of about 64 kDa.
Preferably, the purified human LSR receptor of the present invention is characterized in that it contains an xcex1 subunit comprising the amino acid sequence of SEQ ID 8 or a sequence homologous thereto, or an xcex1xe2x80x2 subunit comprising the amino acid sequence of SEQ ID 10 or a sequence homologous thereto, and one, preferably three, xcex2 subunits comprising the amino acid sequence of SEQ ID 12 or a sequence homologous thereto.
A particularly preferred embodiment of the LSR receptors of the present invention is a recombinant LSR receptor obtained by expressing, in a recombinant host, one or more nucleotide sequences according to the invention. This preferred recombinant receptor consists of an xcex1 or xcex1xe2x80x2 subunit and one, preferably three, xcex2 subunits, in particular an xcex1 or xcex1xe2x80x2 subunit and three xcex2 subunits of a human LSR receptor.
The invention relates to polypeptides, characterized in that they are a constituent of an LSR receptor according to the invention.
It should be understood that the invention does not relate to the polypeptides in a natural form, that is to say that they are not taken in their natural environment. Indeed, the invention relates to the peptides obtained by purification from natural sources, or alternatively obtained by genetic recombination, or alternatively by chemical synthesis, and capable, in this case, of containing non-natural amino acids, as will be described below. The production of a recombinant polypeptide, which may be carried out using one of the nucleotide sequences according to the invention or a fragment of one of these sequences, is particularly advantageous because it makes it possible to obtain an increased level of purity of the desired polypeptide.
The invention therefore relates to a purified, isolated or recombinant polypeptide comprising a sequence of at least 5, preferably at least 10 to 15, consecutive amino acids of an LSR receptor, as well as the homologues, equivalents or variants of the said polypeptide, or one of their fragments. Preferably, the sequence of at least 10 to 15 amino acids of the LSR receptor is a biologically active fragment of an LSR receptor.
More particularly, the invention relates to purified, isolated or recombinant polypeptides comprising a sequence of at least 10 to 15 amino acids of a rat LSR receptor, of a mouse LSR receptor or of a human LSR receptor.
In the present description, the term polypeptide will be used to also designate a protein or a peptide.
The subject of the present invention is also purified nucleic acid sequences, characterized in that they encode an LSR receptor or a polypeptide according to the invention.
The invention relates to a purified nucleic acid, characterized in that it comprises at least 8, preferably at least 10 and more particularly at least 15 consecutive nucleotides of the polynucleotide of a genomic, cDNA or RNA sequence of the LSR receptor, as well as the nucleic acid sequences complementary to this nucleic acid.
More particularly, the invention relates to the purified, isolated or recombinant nucleic acids comprising a sequence of at least 8, preferably at least 10 and more particularly at least 15 consecutive nucleotides of the polynucleotide of a nucleic sequence of a mouse LSR receptor or of a human LSR receptor.
The invention also relates to the variant, mutated, equivalent or homologous nucleic sequences of the nucleic sequences according to the invention, or one of their fragments. It finally relates to the sequences capable of hybridizing specifically with the nucleic sequences according to the invention.
The invention therefore also relates to the nucleic acid sequences contained in the gene encoding the LSR receptor, in particular each of the exons of the said gene or a combination of exons of the said gene, or alternatively a polynucleotide extending over a portion of one or more exons. Preferably, these nucleic acids encode one or more biologically active fragments of the human LSR receptor.
The present invention also relates to the purified nucleic acid sequences encoding one or more elements for regulating the expression of the LSR gene. Also included in the invention are the nucleic acid sequences of the promoter and/or regulator of the gene encoding the receptor according to the invention, or one of their allelic variants, the mutated, equivalent or homologous sequences, or one of their fragments.
The invention also relates to the purified nucleic sequences for hybridization comprising at least 8 nucleotides, characterized in that they can hybridize specifically with a nucleic sequence according to the invention.
Preferably, nucleic acid fragments or oligonucleotides, having as sequences the nucleotide sequences according to the invention can be used as probes or primers.
The invention also comprises methods for screening cDNA and genomic DNA libraries, for the cloning of the isolated cDNAs and/or the genes coding for the receptor according to the invention, and for their promoters and/or regulators, characterized in that they use a nucleic sequence according to the invention.
The nucleic sequences, characterized in that they are capable of being obtained by one of the preceding methods according to the invention or the sequences capable of hybridizing with the said sequences, form part of the invention.
The invention also comprises the cloning and/or expression vectors containing a nucleic acid sequence according to the invention.
The vectors according to the invention, characterized in that they comprise elements allowing the expression and/or the secretion of the said sequences in a host cell, also form part of the invention.
The invention comprises, in addition, the host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention, as well as the mammals, except man, comprising one of the said transformed cells according to the invention.
Among the mammals according to the invention, there will be preferred animals such as mice, rats or rabbits, expressing a polypeptide according to the invention, the phenotype corresponding to the normal or variant LSR receptor, in particular mutated of human origin.
These cells and animals can be used in a method of producing a recombinant polypeptide according to the invention and can also serve as a model for analysis and screening.
The invention also relates to the use of a cell, of a mammal or of a polypeptide according to the invention for studying the expression and the activity of the receptor according to the invention, and the direct or indirect interactions between the said receptor and chemical or biochemical compounds which may be involved in the activity of the said receptor.
The invention also relates to the use of a cell, of a mammal or of a polypeptide according to the invention for screening a chemical or biochemical compound capable of interacting directly or indirectly with the receptor according to the invention, and/or capable of modulating the expression or the activity of the said receptor.
The invention also relates to the synthesis of synthetic or recombinant polypeptides of the invention, in particular by chemical synthesis or using a nucleic acid sequence according to the invention.
The polypeptides obtained by chemical synthesis and capable of comprising non-natural amino acids corresponding to the said recombinant polypeptides are also included in the invention.
The method of producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transformed cells are cultured under conditions allowing the expression of a recombinant polypeptide having a polypeptide sequence according to the invention, and in that the said recombinant polypeptide is recovered.
The recombinant polypeptides, characterized in that they are capable of being obtained by the said method of production, also form part of the invention.
The mono- or polyclonal antibodies or fragments thereof, chimeric or immunoconjugated antibodies, characterized in that they are capable of specifically recognizing a polypeptide or a receptor according to the invention, form part of the invention.
There may be noted in particular the advantage of antibodies specifically recognizing certain polypeptides, variants or fragments, which are in particular biologically active, according to the invention.
The invention also relates to methods for the detection and/or purification of a polypeptide according to the invention, characterized in that they use an antibody according to the invention.
The invention comprises, in addition, purified polypeptides, characterized in that they are obtained by a method according to the invention.
Moreover, in addition to their use for the purification of polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, may also be used for the detection of these polypeptides in a biological sample.
More generally, the antibodies of the invention may be advantageously used in any situation where the expression, normal or abnormal, of a polypeptide of the LSR receptor, normal or mutated, needs to be observed.
Also forming part of the invention are the methods for the determination of an allelic variability, a mutation, a deletion, a loss of heterozygosity or a genetic abnormality, characterized in that they use a nucleic acid sequence or an antibody according to the invention.
These methods relate to, for example, the methods for the diagnosis of the predisposition to obesity, to the associated risks, or to pathologies associated with abnormalities in the metabolism of cytokines, by determining, in a biological sample from the patient, the presence of mutations in at least one of the sequences described above. The nucleic acid sequences analysed may be either the genomic DNA, the cDNA or the mRNA.
Nucleic acids or antibodies based on the present invention can also be used to allow a positive and differential diagnosis in a patient taken in isolation, or a pre-symptomatic diagnosis in an at risk subject, in particular with a familial history.
In addition, the detection of a specific mutation may allow an evolutive diagnosis, in particular as regards the intensity of the pathology or the probable period of its appearance.
Also included in the invention are the methods for selecting chemical or biochemical compounds capable of interacting, directly or indirectly, with the receptor or the polypeptide or nucleotide sequences according to the invention, and/or allowing the expression or the activity of the LSR receptor to be modulated.
The invention relates in particular to a method for selecting chemical or biochemical compounds capable of interacting with a nucleic acid sequence contained in a gene encoding an LSR receptor, the said method being characterized in that it comprises bringing a host cell expressing an LSR receptor or a fragment of the said receptor into contact with a candidate compound capable of modifying the expression or the regulation of the expression of the said nucleic sequence, and detecting, directly or indirectly, a modification of the expression or of the activity of the LSR receptor.
The invention also relates to a method for selecting chemical or biochemical compounds capable of interacting with the LSR receptor, the said method being characterized in that it comprises bringing an LSR receptor or a fragment of the said receptor, or a host cell expressing an LSR receptor or a fragment of the said receptor, into contact with a candidate compound capable of modifying the LSR activity, and detecting, directly or indirectly, a modification of the activity of the LSR receptor or the formation of a complex between the candidate compound and the said LSR receptor or the said polypeptide.
The invention comprises the compounds capable of interacting directly or indirectly with an LSR receptor as well as the compounds capable of interacting with one or more nucleic sequences of the LSR receptor. It also comprises the chemical or biochemical compounds allowing the expression or the activity of the receptor according to the invention to be modulated. The compounds, characterized in that they were selected by one of the methods according to the present invention, also form part of the invention.
In particular, among these compounds according to the invention, there are preferred the antibodies according to the invention, the polypeptides according to the invention, the nucleic acids, oligonucleotides and vectors according to the invention, or a leptin or one of its derived compounds, preferably one of its protein variants, or leptins which are chemically modified or are obtained by genetic recombination, or the protein gC1qR or one of its analogues, or one of their fragments.
The invention comprises, finally, compounds capable of modulating the expression or the activity of the receptor according to the invention, as medicament for the prevention of pathologies and/or of pathogeneses such as obesity and anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, and more generally the various pathologies associated with abnormalities in the metabolism of cytokines.
The invention relates to a purified LSR receptor ( less than  less than Lipolysis Stimulated Receptor greater than  greater than ), preferably hepatic, consisting of at least one xcex1 or xcex1xe2x80x2 subunit and at least one xcex2 subunit. The xcex1 subunit has a molecular weight of about 66 kDa in rats and in mice and of about 72 kDa in humans. The xcex1xe2x80x2 subunit has a molecular weight of about 64 kDa in rats and in mice and of about 70 kDa in humans. The xcex2 subunit has a molecular weight of about 58 kDa in rats and in mice and of about 64 kDa in humans.
The inventors have formulated the hypothesis according to which the most abundant, and probably the most active, form of the LSR receptor is that in which an xcex1 or xcex1xe2x80x2 subunit and three xcex2 subunits exist. It appears, however, possible that the xcex1 and xcex1xe2x80x2 subunits, on the one hand, and the xcex2 subunit, on the other, have distinct biological functions and that these functions can be performed in a cell independently of their assembly in the form of a receptor.
The inventors have also observed that a complex can form between the LSR receptor and the gC1qR receptor having a molecular weight of about 33 kDa, or a homologous protein. It appears that the gC1qR receptor is transiently combined with the LSR receptor and that the presence of a C1q protein or of homologous proteins makes it possible not only to dissociate gC1qR from the LSR receptor but also to activate the LSR receptor, including in the absence of fatty acids.
The present invention therefore relates to a receptor, in particular of hepatic cells, characterized in that it is capable, in the presence of free fatty acids, of binding lipoproteins, and in the absence of free fatty acids, of binding a cytokine, preferably the bound leptin, lipoproteins and cytokine being incorporated and then degraded by the cell, it being possible for the said receptor, in addition, to bind the gC1qR protein or one of its analogous proteins.
The LSR receptor represents the principal pathway for the elimination of lipoproteins of intestinal origin and of particles high in triglycerides, in particular VLDLs and chylomicrons. The LSR receptor can also serve as a pathway for the elimination of LDLs, particles high in cholesterol, which are for the most part removed by the LDL receptor pathway, but of which about 30% are eliminated at the hepatic level by pathways different from the LDL receptor.
The inventors have in fact demonstrated that the LSR receptor is capable of binding lipoproteins, in particular the lipoproteins high in triglycerides, and then of internalizing and degrading them. This lipoprotein clearance activity by the receptor requires the presence of free fatty acids, for example oleate, and is inhibited in the presence of antibodies directed against LSR or against peptides derived from LSR.
The inventors have also demonstrated that in the absence of free fatty acids, for example oleate, the LSR receptor is capable of binding cytokines, preferably leptin. The leptin clearance function is, however, only possible if the receptor has not bound fatty acids produced by the hepatic lipase or by the hormone-sensitive lipase of the adipose tissue. Once the cytokines have been bound, the LSR receptor internalizes them and degrades them. This cytokine, preferably leptin, degradation activity is inhibited by antibodies directed against LSR or against peptides derived from LSR.
The inventors have shown that it is the a subunit of the LSR receptor which is most particularly involved in the binding of cytokines, and preferably of leptin.
Furthermore, the inventors have shown, with the aid of mice, that, in vivo, the LSR receptors carry out the hepatic capturing of cytokines, preferably of leptin.
The high levels of leptin in all obese human subjects can be explained by several molecular mechanisms which are capable of reducing the hepatic clearance of leptin, including in particular:
a) alteration of one or more genes for LSR, and/or of their promoters
b) facilitation, by post-transcriptional modifications, of the allosteric rearrangement allowing the passage from the cytokine-competent conformation to the lipoprotein receptor conformation;
c) deficiency in the transport of vesicles containing LSR from, or towards, the plasma membrane (this function depends on the integrity of the cytoskeleton)
d) increase in the degradation of LSR;
e) increase in the lipid calorie ration which, by diverting the receptor towards the clearance of lipoproteins, reduces in part its capacity to degrade leptin.
Finally, the inventors have demonstrated that cytokines, preferably leptin, modulate the activity of the LSR receptor in the presence of free fatty acids. More particularly, the cytokines increase the lipoprotein clearance activity of the LSR receptor and more precisely, the binding, internalization and degradation of the VLDLs and LDLs. This increase in the LSR activity could be the result of the increase in the apparent number of LSR receptor at the surface of the cells following an increase in protein synthesis and following a mobilization of endocytosis vesicles. In addition, the inventors have shown, with the aid of mice, that, in vivo, cytokines, preferably leptin, are capable of reducing postprandial lipaemic response.
Leptin, and probably other cytokines, are therefore regulators of the activity of LSR. A syndrome of resistance to leptin, or to other cytokines, can lead to a hypertriglyceridemia, which is either permanent or limited to the postprandial phase.
The role played by LSR in the clearance of leptin makes it possible to formulate a physiopathological model which requires a revision of the strategies used for treating obesity. It is indeed essential to reduce the concentrations of leptin in obese human subjects in order to restore the physiological fluctuations of this hormone.
Accordingly, it is possible to envisage using compounds for the treatment of obesity allowing modulation of the number of LSR receptors, of their recycling rate, or of the change in their conformation, and/or allowing in particular:
1. leptinemia, and therefore the sensations of satiety and of hunger, to be controlled;
2. normal leptin concentrations to be restored and normal regulation of dietary habit by the normal perception of the sensations of hunger and of satiety;
3. triglyceridemia to be controlled;
4. the plasma concentrations of residues of chylomicrons, highly atherogenic particles, to be regulated.
The role played by the LSR receptor in the hepatic clearance of lipoproteins of intestinal region makes it possible to envisage using compounds capable of modulating the expression and/or the activity of LSR in order to modulate the distribution of lipids of dietary origin between the peripheral tissues, in particualr the adipose tissues, and the liver. A treatment of obesity will consist in promoting the hepatic degradation of lipoproteins, and thereby reducing their storage in the adipose tissue, and regulating their plasma concentrations. The latter effect makes it possible to envisage the use of such compounds to reduce the risks associated with obesity, in particular the atherogenic risks.
It is possible to envisage using methods of regulating the activities of LSR to introduce treatments which make it possibile to overcome the vicious circle which characterizes anorexia nervosa. By reducing the number of receptors, it should be possible to promote weight gain in anorexic or undernourished subjects.
Under these conditions, it is advantageous to selectively inhibit the clearance of leptin by using synthetic peptides or pharmacological molecules which either reduce the synthesis of LSR or block its capacity to bind leptin and/or lipoproteins, or alternatively increase the catabolism of the receptor.
Analysis of the primary structure of the xcex1 subunit of LSR, as described below, shows a site homologous to the cytokine binding sites present on their receptors, as well as two routing signals which allow endocytosis and rapid degradation of ligands in the lysozomes. This observation is new in the sense that the cytokine receptors do not allow the internalization and the degradation of ligands. These receptors have been characterized on the basis of their intracellular signalling properties.
Thus, in addition to it having the property of allowing the proteolytic degradation of lipoproteins and of leptin, it is highly probable that the LSR receptor also carries out the degradation of other cytokines. This function can be studied by virtue of the anti-LSR antibodies and of transfected CHO cells expressing the xcex1 subunit of LSR as described in Example 4. The involvement of LSR in the clearance of cytokines is essential because these molecules play an important role in the regulation of the metabolism of lipids, of the metabolism of glucose, and in the regulation of food intake and of weight gain.
The molecular mechanisms by which the cytokines modulate the physiological functions involved in obesity and its complications are numerous and complex. It is worth noting, however, the fact that abnormalities in the metabolism of cytokines are associated with hypertriglyceridemia which frequently accompanies viral, bacterial or protozoal infections. Moreover, cytokines, and more particularly Tumor Necrosis Factor (TNF), induce a transient hypertriglyceridemia similar to that observed in certain forms of obesity-related diabetes.
The reduction in the number of LSR receptors expressed in the liver of obese mice could explain a deficiency in the elimination of some cytokines, this deficiency causing metabolic disruptions such as those found in obesity. The use of hepatic cells in culture, and of the various models of obese animals cited below, will make it possible to determine, among all the cytokines and more particularly those which induce weight loss (IL-6, LIF, OSM, CNTF, IL-11, IL-12xcex1, as well as TNFxcex1 and TNFxcex2), those which modulate the expression and/or the activity of LSR. The determination of such cytokines can, for example, be carried out using methods such as those presented in Examples 4 to 6.
Finally, analysis of the primary structure of the xcex1 LSR reveals potential phosphorylation sites. This opens the perspective of a regulation of cellular activity by the LSR receptor. A particularly important example would be the involvement of LSR in the regulation of the production of  less than  less than Acute Phase Proteins greater than  greater than  under the impetus of various stimuli, including cytokines.
The involvement of LSR in the clearance and the degradation of cytokines may, in addition, not be limited to the liver. Indeed, while it has been demonstrated that the expression of LSR is predominantly hepatic, it is also certain that the expression of this receptor is not limited to this organ. Preliminary Northern-blot analysis on various human tissues has been able to reveal, in addition to the hepatic products, expression products in the kidney and in the testicle. A more thorough analysis will make it possible to show the different tissues expressing LSR in humans. In this perspective, LSR could be involved in the degradation of cytokines not only at the hepatic level, but also at the level of the peripheral tissues. A deficiency in this activity could be involved in the pathogenesis of autoimmune diseases, of multiple sclerosis and of rheumatoid arthritis. Accumulation of cytokines is frequently found in the pathogenesis of these diseases.
The invention relates to polypeptides, characterized in that they are a constituent of an LSR receptor according to the invention. The invention relates more particularly to the polypeptides characterized in that they constitute the xcex1, xcex1xe2x80x2 or xcex2 subunits of the LSR receptor.
The invention relates more particularly to a purified, isolated or recombinant polypeptide comprising a sequence of at least 5, preferably of at least 10 to 15 consecutive amino acids of an LSR receptor, as well as the homologues, equivalents or variants of the said polypeptide, or one of their fragments. Preferably, the sequence of at least 10 to 15 amino acids of the LSR receptor is a biologically active fragment of an LSR receptor.
Preferably, the invention relates to purified, isolated or recombinant polypeptides comprising a sequence of at least 10 to 15 amino acids of a rat LSR receptor, of a mouse LSR receptor or of a human LSR receptor.
In a first preferred embodiment of the invention, the polypeptide is characterized in that it comprises a sequence of at least 10 to 15 consecutive amino acids of a sequence chosen from the group comprising the sequences of SEQ ID 2, SEQ ID 4 and SEQ ID 6, as well as the variants, equivalents or homologues of this polypeptide, or one of their fragments. Preferably, the polypeptide is a homologue or a biologically active fragment of one of the abovementioned sequences.
In a second preferred embodiment of the invention, the polypeptide is characterized in that it comprises a sequence of at least 10 to 15 consecutive amino acids of a sequence chosen from the group comprising the sequences of SEQ ID 16, SEQ ID 17 and SEQ ID 18, as well as the variants, equivalents or homologues of this polypeptide, or one of their fragments. Preferably, the polypeptide is a homologue or a biologically active fragment of one of the abovementioned sequences.
In a third preferred embodiment of the invention, the polypeptide is characterized in that it comprises a sequence of at least 10 to 15 consecutive amino acids of a sequence chosen from the group comprising the sequences of SEQ ID 8, SEQ ID 10 and SEQ ID 12, as well as the variants, equivalents or homologues of this polypeptide, or one of their fragments. Preferably, the polypeptide is a homologue or a biologically active fragment of one of the abovementioned sequences.
Among the preferred polypeptides of the invention, there will be noted particularly the polypeptides having the human sequence SEQ ID 8, SEQ ID 10 or SEQ ID 12, as well as those having the rat sequence SEQ ID 2, SEQ ID 4 or SEQ ID 6, or those having the mouse sequence SEQ ID 16, SEQ ID 17 or SEQ ID 18. The fragments corresponding to the domains represented in FIGS. 1 to 6, whose positions on the sequences corresponding to SEQ ID 2, 8 or 16, are indicated in Tables 1, 3 and 4.
Finally, the invention also relates to the polypeptides of SEQ ID 29 and SEQ ID 30.
The present invention also relates to polypeptides comprising the polypeptides described above, as well as their homologous, equivalent or variant polypeptides, as well as the fragments, preferably biologically active, of the said polypeptides.
Among the polypeptides according to the invention, also preferred are the polypeptides comprising or consisting of an amino acid sequence chosen from the amino acid sequences as described above, characterized in that the said polypeptides are a constituent of the receptor according to the invention.
The systematic analysis of the products of the 3 rat cDNAs described in the present application is schematically represented in FIG. 1. The xcex1 subunit of the rat LSR receptor, a protein encoded by the longer cDNA (LSR-Rn-2097), has the following characteristics.
Potential glycosylation sites are found at positions 12-14 and 577-579. A potential site of attachment of glycosaminoglycans is found at position 14-17.
Several phosphorylation sites are located at the level of the NH2-terminal end (positions 193-196, 597-600, 169-171, 172-174, 401-403, 424-426, 464-466, 467-469, 185-188, 222-225, 436-439, 396-399, 504-507, 530-533, 624-627, 608-615), suggesting that the latter is oriented towards the intracellular region.
Moreover, the protein has, on the NH2-terminal side, a hydrophobic amino acid sequence separated into two parts by 2 amino acids inducing a hairpin structure in which the two arms would consist of hydrophobic amino acids. It is reasonable to assume that this region represents the fatty acid binding site of LSR. The glove-finger structure thus produced can accommodate an aliphatic hydrocarbon chain. The two amino acids are, more precisely in the case of rat LSR, two Prolines situated at positions 31 and 33 of the polypeptide sequence of the xcex1 subunit.
Still on the NH2-terminal side is a consensus sequence for binding to clathrin, a protein which lawns the inner surface of the  less than  less than coated pits greater than  greater than  (Chen et al., 1990). These specific regions of the plasma membrane allow rapid endocytosis of membrane proteins. Such a consensus sequence is found at the level of the LRP-xcex12-macroglobulin receptor, of CRAM and of the LDL receptor (Herz et al., 1988; Lee et al., 1990; Goldstein et al., 1995). The consequence of a mutation at this level is a substantial delay in the internalization of the LDLs and induces familial hypercholesterolemia (Davis et al., 1986).
The receptor then possesses a hydrophobic amino acid sequence which constitutes a potential transmembrane domain. The length of this segment allows only one passage across the phospholipid bilayer (Brendel et al., 1992).
Between this clathrin binding signal and the hydrophobic chain corresponding to the single transmembrane segment are 2 motifs LI et LL (Letourneur et al., 1992). These two motifs are found in the following proteins: glut 4 glucose carrier (Verhey et al., 1994); the nonvariant chain and the histocompatibility complex class II (Zhong et al., 1997; Parra-Lopez et al., 1997). These signals control endocytosis and the intracellular addressing of proteins in the peripheral membrane system.
On the C-terminal side, there is then a cysteine-rich region which exhibits homology with the cytokine receptors and more particularly: the TNF 1 and 2 (Tumor Necrosis Factor 1 and 2) receptors; the low-affinity NGF (Nerve Growth factor) receptor; the Shope fibroma virus TNF soluble receptor; CD40, CD27 and CD30, receptors for the cytokines CD40L, CD27L and CD30L; the T cell protein 4-1BB, receptor for the putative cytokine 4-1BBL, the FAS antigen (APO 1), receptor for the FASL protein involved in apoptosis, the T cell OX40 antigen, receptor for the cytokine OX40L, and the vaccinia virus A53 protein (Cytokines and their receptors, 1996; Banner et al., 1993).
In addition to this cysteine-rich segment, there is a region of amino acids which are alternately charged + and xe2x88x92 (Brendel et al., 1992). This region provides a potential binding site for the apoprotein ligands Apo B and Apo E.
This region contains, in addition, an RSRS motif found in lamin (Simos et al., 1994) and in SF2xe2x80x2 (Krainer et al., 1991).
The LSR xcex1xe2x80x2 form encoded by the LSR-Rn-2040 cDNA possesses all the domains described above based on the LSR a sequence encoded by the LSR-Rn-2097 cDNA, with the exception of the LI/LL element, whose Leucine doublet is removed by alternative splicing. Although possessing sequences which are very similar, the subunits a encoded by LSR-Rn-2097 and xcex1xe2x80x2 encoded by LSR-Rn-2040 could therefore differ in their recycling rate and their addressing. The xcex2 form encoded by LSR-Rn-1893 does not possess a transmembrane domain or a region rich in cysteines and homologous to the cytokine receptors. However, it possesses at the NH2-terminal level the hydrophobic region separated by a repetition of prolines, the region rich in charged amino acids and the RSRS motif. This constituent is probably positioned entirely outside the cell where it is bound via disulphide bridges either to the product of LSR-Rn-2040, or to that of LSR-Rn-2097.
Table 1 below lists the different domains or motifs described above, indicates whether or not they belong to each of the subunits of the LSR receptor, as well as the positions of the start and end of the said domains or motifs, or of regions carrying the said domains or motifs, as indicated in the sequence of SEQ ID 2.
The lengths of the polypeptide sequences, as well as the SEQ IDs of their respective sequences in the listing included, of the three types of subunit of the LSR receptors according to the invention, in rats, mice and humans, are indicated in Table 2a below.
These polypeptide sequences were obtained from each of the three corresponding cDNA sequences, in rats, mice and humans, which will be described in detail later. These polypeptide sequences were obtained from each of the three corresponding cDNA sequences, in rats, mice and humans, which will be described in detail later. The nomenclature used to designate these cDNA sequences, which reflects their length in terms of nucleotides, as well as the SEQ IDs of their respective sequences in the listing included, are presented in Table 2b below.
The protein sequence, corresponding to the a subunit of the LSR receptor, deduced from the LSR-Hs-2062 sequence has a length of 649 amino acids. It is aligned with the protein sequences deduced from LSR-Mm-1886, 594 amino acids long, and from LSR-Rn-2097, 593 amino acids long (FIGS. 2A and 2B). The conservation of the protein sequences is very high (respectively 80.2% and 82.2% identity for 591 and 590 overlapping amino acids). The functional domains identified in the protein sequence of the rat LSR a are found in the human LSR a sequence as well as in that of the murine LSR a (FIGS. 2A and 2B).
The human proteins corresponding to the LSR-Hs-2005 (xcex1xe2x80x2) and LSR-Hs-1858 (xcex2) forms have a predicted size of 630 and 581 amino acids respectively. The rat proteins corresponding to the LSR-Rn-2040 (xcex1xe2x80x2) and LSR-Rn-1893 (xcex2) forms have a predicted size of 574 and 525 amino acids respectively. The mouse proteins corresponding to the LSR-Mm-1829 (xcex1xe2x80x2) and LSR-Mm-1682 (xcex2) forms have a predicted size of 575 and 526 amino acids respectively. The alignment of the three human forms (FIGS. 3A and 3B), of the three forms described in rats (FIGS. 4A and 4B) and of the three forms described in mice (FIGS. 5A and 5B) shows that in the three species, all the protein forms conserve the NPGY signal for binding to clathrin and the RSRS motif. The human (product of LSR-Hs-2062), rat (product of LSR-Rn-2097) and mouse (product of LSR-Mm-1886) long forms (xcex1) exhibit all the functional characteristics of LSR. The three short forms (xcex2) (respective products of LSR-Hs-1817, LSR-Rn-1893 and LSR-Mm-1682) lose the di-leucine domain for lysosomal addressing, the transmembrane domain and the cytokine receptor signature. It is also possible to observe that the three intermediate forms (xcex1xe2x80x2) (product of LSR-Hs-2005, of LSR-Rn-2040 and LSR-Mn-1829) lose the di-leucin domain, the transmembrane domain and the domain corresponding to the cytokine receptor signature being conserved (FIGS. 3A, 3B, 4A, 4B, 5A and 5B). FIG. 6 finally represents the proteins derived from the three cDNA forms identified in humans, and the motifs carried by each of them as a result of the splicing from which each is derived.
Table 3 below lists the different domains or motifs described above, as well as the positions of the start and end of the said domains or motifs, or of regions carrying the said domains or motifs, as indicated in the mouse SEQ ID 16 sequence.
Table 4 below lists the different domains or motifs described above, as well as the positions of the start and end of the said domains or motifs, or of regions carrying the said domains or motifs, as indicated in the human SEQ ID 8 sequence.
In conclusion, the similarity in the sequence and structure of LSR which is described above makes it possible to extrapolate to humans the observations made in rats and/or mice.
Homologous polypeptide will be understood to mean the polypeptides exhibiting, compared with the natural polypeptide, certain modifications such as in particular a deletion, truncation, extension, chimeric fusion and/or mutation, in particular a point mutation. Among the homologous polypeptides, those in which the amino acid sequence exhibits at least 80%, preferably 90%, homology with the amino acid sequences of the polypeptides according to the invention are preferred.
Equivalent polypeptide will be understood to mean a polypeptide having at least one of the activities of the LSR receptor, in particular the activity of the receptor for lipoproteins or chylomicrons, the activity of the receptor for cytokine, in particular leptin, or the activity of the receptor for the gC1q-R protein or one of its analogous proteins. Equivalent polypeptide will also be understood to mean any polypeptide resulting from the alternative splicing of the genomic nucleic sequence encoding the polypeptides according to the invention.
Variant polypeptide (or protein variant) will be understood to mean all the mutated polypeptides which may exist, in particular in human beings, and which correspond in particular to truncations, deletions and/or additions of amino acid residues, substitutions or mutations, in particular point mutations, as well as the artificial variant polypeptides which will nevertheless be called variant polypeptides. In the present case, the variant polypeptides will be in particular partly associated with the onset and with the development of obesity or anorexia. They may also be associated with the onset and/or development of the risks or complications associated with obesity, in particular at the cardiovascular level, and/or of pathologies associated with abnormalities in the metabolism of cytokines.
Polypeptide fragment is understood to mean a polypeptide or a peptide encoded by a nucleic sequence comprising a minimum of 15 nucleotides or bases, preferably 20 bases or 30 bases. These fragments may comprise in particular a point mutation, compared with the normal polypeptide sequence, or may correspond to specific amino acid sequences of variant polypeptides, artificial or existing in humans, such as those linked to a polymorphism linked in particular to obesity or to the abovementioned pathologies.
Biologically active fragment will be understood to mean in particular a fragment of an amino acid sequence of a polypeptide:
exhibiting at least one of the LSR receptor activities, in particular the lipoprotein receptor activity, or the cytokine, particularly leptin, receptor activity and/or cell signalling activity, and/or
capable of being recognized by an antibody specific for the receptor according to the invention, and/or
capable of being recognized by a compound capable, for example by neutralizing the binding of a ligand specific for the said receptor, of modulating the activity of the LSR receptor, and/or
capable of modulating the addressing and/or cellular location of the LSR receptor, and/or
more generally constituting a biologically active domain or motif of the LSR receptor.
Among the preferred biologically active fragments according to the invention, there are in particular:
the fragments comprising a clathrin binding site,
the fragments comprising a fatty acid binding site, in particular a fatty acid binding site comprising a hydrophobic amino acid sequence separated into two parts by two contiguous prolines, which induce a hairpin structure whose arms consist of hydrophobic amino acids,
the fragments comprising a hydrophobic region constituting a transmembrane domain,
the fragments comprising a region capable of controlling endocytosis and intracellular addressing of the proteins in the peripheral membrane system, in particular a fragment comprising a site containing the LI and LL motifs,
the fragments comprising a cytokine binding site, in particular a site including a cysteine-rich region,
the fragments comprising a region defining a potential binding site for lipoprotein ligands such as ApoB and ApoE, in particular a region comprising a sequence of amino acids alternately charged + and xe2x88x92, and
the fragments comprising an RSRS motif.
There are in particular among these fragments polypeptides as defined in Tables 1, 2 and 4, or any fragments of the nucleotides of SEQ ID 2, 8 or 16, comprising the said polypeptides, and any equivalent, homologous or variant fragments.
Other preferred fragments include antigenic peptides such as those having the sequences SEQ ID 29 and 30.
The subject of the present invention is isolated nucleic acid sequences, characterized in that they encode an LSR receptor or a polypeptide according to the invention.
More particularly, the invention relates to a purified nucleic acid, characterized in that it comprises at least 8, preferably at least 10 and more particularly at least 15 consecutive nucleotides of the polynucleotide of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises at least 8, preferably at least 10 and more particularly at least 15 consecutive nucleotides of the polynucleotide of SEQ ID 41, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid encoding the human LSR receptor, characterized in that it comprises a nucleotide sequence corresponding to nucleotides 1898 to 21094, particularly to nucleotides 2001 to 20979, more particularly to nucleotides 2145 to 20979 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to the nucleic acid sequences contained in the gene encoding the human LSR receptor, in particular each of the exons of the said gene or a combination of exons of the said gene, or alternatively a polynucleotide extending over a portion of one or more exons. Preferably, these nucleic acids encode one or more biologically active fragments of the human LSR receptor.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence corresponding to nucleotides 21095 to 22976 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence chosen from the group comprising the sequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence chosen from the group comprising the sequences of SEQ ID 1, SEQ ID 3 and SEQ ID 5, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence chosen from the group comprising the sequences of SEQ ID 13, SEQ ID 14 and SEQ ID 15, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence corresponding to nucleotides 1898 to 2001 of SEQ ID 19 or preferably to nucleotides 1898 to 2144 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid.
The invention also relates to a purified nucleic acid, characterized in that it comprises a nucleotide sequence corresponding to nucleotides 20980 to 21094 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid.
Among the nucleic acids according to the invention, the nucleic acids having the nucleotide sequence chosen from the group comprising the sequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, the sequences of SEQ ID 1, SEQ ID 3 and SEQ ID 5, as well as the sequences of SEQ ID 13, SEQ ID 14 and SEQ ID 15, as well as their complementary sequences, are preferred.
Also forming part of the invention are the variant, mutated, equivalent or homologous sequences of the sequences according to the invention, as well as their fragments and the nucleic sequences capable of hybridizing specifically with the sequences according to the invention.
The invention therefore relates to the genomic sequence of the human LSR receptor, preferably the sequence of SEQ ID 19, as well as their complementary sequences or one of their allelic variants, the mutated, equivalent or homologous sequences, or one of their fragments.
The gene for human LSR (SEQ ID 19) comprises 10 exons distributed over 21 094 bp. The size of the exons is respectively: 356, 345, 120, 57, 147, 174, 60, 132, 626 and 141 bp (Table 5).
The EXON column indicates the exons numbered from 1 to 10 in the 5xe2x80x2-3xe2x80x2 order of their position on the genomic sequence. The START and END columns indicate respectively the position of the first and of the last nucleotide of the exon considered. The sequences of the splicing site bordering the exon in 5xe2x80x2 and 3xe2x80x2 are indicated in the 5xe2x80x2SPLIC and 3xe2x80x2SPLIC columns. The BL 5xe2x80x2 and BL 3xe2x80x2 columns indicate the number of bases in 5xe2x80x2 and in 3xe2x80x2, respectively, of an exon which will be used in the reading frame of the messenger only after splicing. For example as exon 7 has a free base in 3xe2x80x2, this exon can be joined to the 5xe2x80x2 end of exon 8 which has 2 free bases in 5xe2x80x2. The combination 1 base+2 bases constitutes the codon which was destroyed by the intron in the genomic sequence. Exon 7 may be joined by its 3xe2x80x2 end to any exon having two free bases in 5xe2x80x2; if the new codon created does not correspond to a stop codon, the open reading frame will be conserved.
Exons 1 and 2 as well as 9 and 10 are necessarily co-spliced, thus forming a 5xe2x80x2 block corresponding to exons 1 and 2 and a 3xe2x80x2 block corresponding to exons 9 and 10. The functional minimal messenger, corresponding to the product of these four exons, could therefore have a size of about 1 331 bp. For the other exons, all the possible combinations make it possible to conserve the open reading frame.
The size of the noncoding exons in 5xe2x80x2 could not be determined with precision. Indeed, the rat 5xe2x80x2 UTR sequences are too divergent from those of humans to finalize the analysis of these sequences and to identify the real 5xe2x80x2 end of the human LSR cDNA. This can be carried out by isolating the 5xe2x80x2 end of the human LSR messengers by the 5xe2x80x2 end capture methods developed by the inventors (WO 96/34981). The polyadenylation site described below is the only one which is present before the USF2 gene, situated in 3xe2x80x2 of the human LSR gene. It is therefore very likely that the untranslated 3xe2x80x2 region of this gene is very short (of an estimated size of about 100 bp). All the sizes given in relation to the human LSR cDNA molecules will therefore have to be adjusted according to the size of the untranslated 5xe2x80x2 end. The human cDNA sequence obtained taking into account all the exons deduced from the analysis of the genomic sequence have a size of 2 158 bp. This form could correspond to the LSR-Rn-2097 form.
The location of some of the signals for expression of the nucleotide sequence of SEQ ID 19 is presented in Table 6 which follows.
The characteristic elements of the messenger RNA molecule are described in the Signal column: Initiation of translation (ATG), termination of translation (STOP) and polyadenylation signal (POLY Ad). The Start and End columns indicate the position as nucleotide for the start and end of these signals on the genomic sequence SEQ ID 19. An ATG signal for initiation of translation is preferred to another because it provides an environment which is more suitable for initiation.
The present invention also relates to the purified nucleic acid sequences encoding one or more elements for regulating the expression of the human LSR gene. Also included in the invention are the nucleic acid sequences of the promoter and/or regulator of the gene encoding the receptor according to the invention, or one of their allelic variants, the mutated, equivalent or homologous sequences, or one of their fragments.
The invention relates more particularly to a purified nucleic acid situated in 5xe2x80x2 of the coding sequence of the LSR gene. This nucleic acid is characterized in that it comprises a nucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid. Shorter fragments of this nucleic acid may also be used as promoters for expression of the LSR gene or of any other sequence encoding a heterologous polypeptide.
The invention also relates to a purified nucleic acid situated in 3xe2x80x2 of the transcribed sequence of the LSR gene. This nucleic acid is characterized in that it comprises a nucleotide sequence corresponding to nucleotides 21095 to 22976 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid. Shorter fragments of this nucleic acid can also be used as elements regulating the expression of genes.
Finally, the invention also relates to the genomic sequence of the human LSR receptor, preferably the sequence of SEQ ID 41, as well as their complementary sequences, or one or their allelic variants, the mutated, equivalent or homologous sequences, or one of their fragments.
It is advantageous to note that a syntheny (conservation of the organization of certain chromosomal regions between species) between the mouse chromosome 7 region where the Lisch7 gene is located, in the immediate vicinity of USF2, and the human chromosome 19 region 19q13, carrying LSR, is well described. The organization of the two Lisch7/LSR and USF2 genes is conserved between species. Likewise, Apo E, which is of a more centromeric location relative to these genes, exists both in mice and in humans. It is remarkable that the LSR lipoprotein receptor and one of their ligands ApoE are located in the same chromosomal region. Indeed, the receptor and the ligand are frequently co-regulated. Such a situation would make it possible to envisage that the phenomena observed in mice are applicable to humans.
The invention relates, in addition, to 3 different cDNAs derived from the LSR receptor gene by alternative splicing. These 3 cDNAs have been identified in humans, rats and mice (Table 2b). They encode the three types of LSR receptor subunits, xcex1 (long), xcex1xe2x80x2 (intermediate) and xcex2 (short). The longest cDNA contains the totality of the 10 exons of the gene. The intermediate cDNA does not comprise exon 4. Finally, the shortest cDNA does not contain exons 4 and 5.
The human LSR-Hs-2062 cDNA nucleotide sequence, encoding the a subunit of the LSR receptor, and the rat LSR-Rn-2097 cDNA nucleotide sequence are 78.6% identical over 1 955 bp which overlap. These figures are respectively 78.8% and 1 851 bp when the murine LSR-Mm-1886 sequence (long form) is aligned with the human sequence. This reflects a very high conservation of the nucleic sequences between species. The highest divergence levels are observed in the untranslated 5xe2x80x2 end (when the sequence is available), in the first coding exon and in the untranslated 3xe2x80x2 end (FIGS. 7A, 7B, 7C, 7D and 7E).
The invention therefore also relates to a purified nucleic acid, characterized in that it is chosen from the group comprising the sequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, the sequences SEQ ID 1, SEQ ID 3 and SEQ ID 5, and the sequences SEQ ID 13, SEQ ID 14 and SEQ ID 15, as well as the nucleic acid sequences complementary to this nucleic acid, or one of their allelic variants, the mutated, equivalent or homologous sequences, or one of their fragments.
The nucleic acids constituting the coding frames of the abovementioned nucleic acids, between the codons for initiation and for termination of translation, also form part of the invention.
The nucleic acids encoding the polypeptide fragments according to the invention are also part of the invention. It will be particularly noted [lacuna] the nucleic acids encode the fragments described in Tables 1, 3 and 4.
Thus, Table 7 describes the position of such nucleic acid fragments on the human sequence of SEQ ID 7.
The invention also relates to a purified nucleic acid corresponding to the sequence of the 5xe2x80x2UTR of the cDNAs encoding the human LSR receptor. This nucleic acid is characterized in that it comprises a nucleotide sequence corresponding to nucleotides 1898 to 2001 of SEQ ID 19 or preferably to nucleotides 1898 to 2144 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid. Shorter fragments of this nucleic acid can also be used.
The invention also relates to a purified nucleic acid corresponding to the sequence of the 3xe2x80x2UTR of the cDNAs encoding the LSR receptor. This nucleic acid is characterized in that it comprises a nucleotide sequence corresponding to nucleotides 20980 to 21094 of SEQ ID 19, as well as the nucleic acid sequences complementary to this nucleic acid. Shorter fragments of this nucleic acid can also be used.
The invention also relates to the purified nucleic acids corresponding respectively to the sequences of the 5xe2x80x2UTR or of the 3xe2x80x2UTR of the cDNAs encoding the rat or mouse LSR receptor. Shorter fragments of this nucleic acid can also be used.
The 5xe2x80x2UTR and 3xe2x80x2UTR may contain elements ( less than  less than responsive elements greater than  greater than  and  less than  less than enhancers greater than  greater than ) which are involved in the regulation of transcription and of translation. These regions have in particular a role in the stability of the mRNAs. Furthermore, the 5xe2x80x2UTR comprises the Shine-Delgarno motif which is essential for the translation of the mRNA.
Nucleic acid, nucleic sequence or nucleic acid sequence are understood to mean an isolated natural, or a synthetic, DNA and/or RNA fragment comprising, or otherwise, non-natural nucleotides, designating a precise succession of nucleotides, modified or otherwise, allowing a fragment, a segment or a region of a nucleic acid to be defined.
Equivalent nucleic sequences are understood to mean nucleic sequences encoding the polypeptides according to the invention taking into account the degeneracy of the genetic code, the complementary DNA sequences and the corresponding RNA sequences, as well as the nucleic sequences encoding the equivalent polypeptides.
Homologous nucleic sequences are understood to mean the nucleic sequences encoding the homologous polypeptides and/or the nucleic sequences exhibiting a level of homology of at least 80%, preferably 90%. According to the invention, the homology is only of the statistical type, which means that the sequences have a minimum of 80%, preferably 90%, of nucleotides in common. They are preferably sequences capable of hybridizing specifically with a sequence of the invention. Preferably, the specific hybridization conditions will be like those found in the examples, or such that they ensure at least 95% homology.
The length of these nucleic sequences for hybridization can vary from 8, 10, 15, 20 or 30 to 200 nucleotides, particularly from 20 to 50 nucleotides, more particularly from 20 to 30 nucleotides.
Allele or allelic variant will be understood to mean the natural mutated sequences corresponding to polymorphisms present in human beings and, in particular, to polymorphisms which can lead to the onset and/or to the development of obesity or of anorexia. These polymorphisms can also lead to the onset and/or to the development of risks or complications associated with obesity, in particular at the cardiovascular level, and/or of pathologies associated with abnormalities in the metabolism of cytokines.
Mutated nucleic sequences are understood to mean the nucleic sequences comprising at least one point mutation compared with the normal sequence.
While the sequences according to the invention are in general normal sequences, they are also mutated sequences since they comprise at least one point mutation and preferably at most 10% of mutations compared with the normal sequence.
Preferably, the present invention relates to mutated nucleic sequences in which the point mutations are not silent, that is to say that they lead to a modification of the amino acid encoded in relation to the normal sequence. Still more preferably, these mutations affect amino acids which structure the LSR complex and/or receptor or the corresponding domains and fragments thereof. These mutations may also affect amino acids carried by the regions corresponding to the receptor sites, for lipoproteins or cytokines, in particular leptin, or to sites for binding of cofactors, in particular or free fatty acids, or alternatively to phosphorylation sites. These mutations may also affect the sequences involved in the transport, addressing and membrane anchorage of LSR.
In general, the present invention relates to the normal LSR polypeptides, the mutated LSR polypeptides as well as fragments thereof and to the corresponding DNA and RNA sequences, the LSR polypeptides designating polypeptides of the receptor according to the invention.
According to the invention, the fragments of nucleic sequences may in particular encode domains of receptors and polypeptides possessing a function or a biological activity as defined above, contain domains or regions situated upstream or downstream of the coding sequence and containing elements for regulating the expression of the LSR gene or alternatively possessing a sequence allowing their use as a probe or as a primer in methods of detection, identification or amplification of nucleic sequences. These fragments preferably have a minimum size of 8, of 10 bases, and fragments of 20 bases, and preferably of 30 bases, will be preferred.
Among the nucleic fragments which may be of interest, in particular for diagnosis, there should be mentioned, for example, the genomic intron sequences of the gene for the LSR complex, such as in particular the joining sequences between the introns and the exons, normal or mutated.
The nucleic acid sequences which can be used as sense or antisense oligonucleotides, characterized in that their sequences are chosen from the sequences according to the invention, also form part of the invention.
Among the nucleic acid fragments of interest, there should thus be mentioned, in particular the antisense oligonucleotides, that is to say whose structure ensures, by hybridization with the target sequence, inhibition of the expression of the corresponding product. There should also be mentioned the sense oligonucleotides which, by interaction with the proteins involved in the regulation of the expression of the corresponding product, will induce either inhibition, or activation of this expression.
The sequences carrying mutations which may be involved in the promoter and/or regulatory sequences of the genes for the LSR complex, which may have effects on the expression of the corresponding proteins, in particular on their level of expression, also form part of the preceding sequences according to the invention.
The nucleic sequences which can be used as primer or probe, characterized in that their nucleic sequence is a sequence of the invention, also form part of the invention.
The present invention relates to all the primers which may be deduced from the nucleotide sequences of the invention and which may make it possible to detect the said nucleotide sequences of the invention, in particular the mutated sequences, using in particular a method of amplification such as the PCR method, or a related method.
The present invention relates to all the probes which may be deduced from the nucleotide sequences of the invention, in particular sequences capable of hybridizing with them, and which may make it possible to detect the said nucleotide sequences of the invention, in particular to discriminate between the normal sequences and the mutated sequences.
The invention also relates to the use of a nucleic acid sequence according to the invention as a probe or a primer for the detection and/or the amplification of a nucleic acid sequence according to the invention.
All the probes and primers according to the invention may be labelled by methods well known to persons skilled in the art, in order to obtain a detectable and/or quantifiable signal.
The present invention also relates to the nucleotide sequences which may comprise non-natural nucleotides, in particular sulphur-containing nucleotides, for example, or nucleotides of xcex1 or xcex2 structure.
The present invention relates, of course, to both the DNA and RNA sequences, as well as the sequences which hybridize with them, as well as the corresponding double-stranded DNAs.
In the text which follows, the preceding DNA sequences will be called genes for the LSR complex, whether they are normal or pathologic sequences.
It should be understood that the present invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. They are sequences which have been isolated, that is to say that they have been collected directly or indirectly, for example by copying (cDNA), their environment having been at least partially modified.
Thus, this may also be both cDNA and genomic DNA, partially modified or carried by sequences which are at least partially different from the sequences carrying them naturally.
These sequences may also be termed non-natural.
The invention also comprises methods for screening cDNA and genomic DNA libraries, for the cloning of the isolated cDNAs, and/or the genes coding for the receptor according to the invention, and for their promoters and/or regulators, characterized in that they use a nucleic sequence according to the invention. Among these methods, there may be mentioned in particular:
the screening of cDNA libraries and the cloning of the isolated cDNAs (Sambrook et al., 1989; Suggs et al., 1981; Woo et al., 1979), with the aid of the nucleic sequences according to the invention,
the screening of 5xe2x80x2 end tag libraries (WO 96/34981) for nucleic sequences according to the invention, and thus the isolation of tags allowing the cloning of complete cDNAs and the corresponding promoters from genomic DNA libraries,
the screening of genomic libraries, for example of BACs, (Chumakov et al., 1992; Chumakov et al., 1995) and, optionally, a genetic analysis by FISH (Cherif et al., 1990) with the aid of sequences according to the invention, allowing isolation and chromosomal location, and then the complete sequencing of the genes encoding the LSR receptor.
Also included in the invention is a sequence, in particular a genomic sequence encoding a receptor or a polypeptide according to the invention, or a nucleic acid sequence of a promoter and/or regulator of a gene encoding a receptor or a polypeptide according to the invention, or one of their allelic variants, a mutated, equivalent or homologous sequence, or one of their fragments, characterized in that it is capable of being obtained by one of the preceding methods according to the invention, or a sequence capable of hybridizing with the said sequences.
The invention also comprises the cloning and/or expression vectors containing a nucleic acid sequence according to the invention.
The vectors according to the invention, characterized in that they comprise the elements allowing the expression and/or the secretion of the said sequences in a host cell, also form part of the invention.
The vectors characterized in that they comprise a promoter and/or regulator sequence according to the invention, or a sequence for cellular addressing according to the invention, or one of their fragments, also form part of the invention.
The said vectors will preferably comprise a promoter, signals for initiation and termination of translation, as well as appropriate regions for regulation of transcription. They must be able to be stably maintained in the cell and may optionally possess particular signals specifying the secretion of the translated protein.
These different control signals are chosen according to the cellular host used. To this end, the nucleic acid sequences according to the invention may be inserted into autonomously replicating vectors inside the chosen host, or integrative vectors of the chosen host.
Among the autonomously replicating systems, there will be preferably used according to the host cell, systems of the plasmid or viral type, it being possible for the viral vectors to be in particular adenoviruses (Perricaudet et al., 1992), retroviruses, poxviruses or herpesviruses (Epstein et al., 1992). Persons skilled in the art know the technologies which can be used for each of these systems.
When the integration of the sequence into the chromosomes of the host cell is desired, it will be possible to use, for example, systems of the plasmid or viral type; such viruses will be, for example, retroviruses (Temin, 1986), or AAVs (Carter, 1993).
Such vectors will be prepared according to the methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods such as, for example, lipofection, electroporation or heat shock.
The invention comprises, in addition, the host cells, in particular eukaryotic and prokaryotic cells, transformed by the vectors according to the invention, as well as transgenic animals, except humans, comprising one of the said transformed cells according to the invention.
Among the cells which can be used for these purposes, there may of course be mentioned bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular mammalian cell cultures (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary cells (CHO), but also insect cells in which it is possible to use methods using baculoviruses, for example (Luckow, 1993). A preferred cellular host for the expression of the proteins of the invention consists of the CHO cells.
Among the mammals according to the invention, there will be preferred animals such as mice, rats or rabbits, expressing a polypeptide according to the invention, the phenotype corresponding to the normal or variant LSR receptor, in particular mutated of human origin.
Among the animal models more particularly of interest here, there are in particular:
transgenic animals exhibiting a deficiency in one of the components of LSR. They are obtained by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of the said chimeras;
transgenic mice overexpressing one or more of the genes for the LSR complex of murine and/or human origin. The mice are obtained by transfection of multiple copies of the genes for the LSR complex under the control of a strong promoter of an ubiquitous nature, or selective for a type of tissue, preferably the liver;
transgenic animals (preferably mice) made deficient in one or more of the genes for the LSR complex, by inactivation with the aid of the LOXP/CRE recombinase system (Rohlmann et al., 1996) or any other system for inactivating the expression of a gene at a precise age of the animal;
animals (preferably rats, rabbits, mice) overexpressing one or more of the genes for the LSR complex, after viral transcription or gene therapy;
crossings of animals deficient in LSR (in particular mice) with animals deficient in, or overexpressing:
the LDL receptor (Herz et al., 1995; Ishibashi et al., 1993)
hepatic lipase (Homanics et al., 1995; Kobayashi et al., 1996)
apoprotein B (Purcellhuynh et al., 1995; Fan et al., 1995)
apoprotein E (Plump et al., 1992; Zhang et al., 1992; Huang et al., 1996)
apoCIII (Aalto-Setxc3xa4lxc3xa4 et al., 1992; Ito et al., 1990; Maeda et al., 1994).
The production of transgenic animals, and the viral or nonviral transfections will be preferably carried out on the following rat and mouse lines:
Zucker rat (fa/fa) (Iida et al., 1996)
AKR/J mouse (West et al., 1992)
ob/ob mouse (Zhang et al., 1994)
ob2j/ob2j mouse (ibid)
tubby mouse (Kleyn et al., 1996; Nobben-Trauth et al., 1996)
fat/fat (Heldin et al., 1995)
agouti mouse (Lu et al., 1994; Manne et al., 1995)
db/db mouse (Chen et al., 1996).
The cells and mammals according to the invention can be used in a method for the production of a polypeptide according to the invention, as described below, and can also serve as a model for analysis and screening.
The transformed cells or mammals as described above can also be used as models so as to study the interactions between the polypeptides of the LSR complex, between these and their partners, chemical or protein compounds, which are involved directly or indirectly in the activities of the receptor for lipoproteins or the receptor for cytokines, and in particular for leptin, and in order to study the different mechanisms and interactions called into play according to the type of activity, or according to whether a normal complex is involved, or a complex in which at least one of the domains is a variant.
In particular, they may be used for the selection of products which interact with the LSR complex, or one of its normal or variant domains, as cofactor or as inhibitor, in particular a competitive inhibitor, or alternatively having an agonist or antagonist activity on the conformational changes in the LSR complex. Preferably, the said transformed cells will be used as a model allowing, in particular, the selection of products which make it possible to combat obesity or the pathologies mentioned above. The said cells may also serve for the detection of the potential risks posed by certain compounds.
The invention also relates to the synthesis of synthetic or recombinant polypeptides of the invention, in particular by chemical synthesis or by the use of a nucleic acid sequence according to the invention.
The polypeptides according to the present invention can be obtained by chemical synthesis using any of the numerous known peptide syntheses, for example the techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by a conventional synthesis in solution.
When the compounds according to the present invention are synthesized by the solid phase method, the C-terminal amino acid is bound to an inert solid support and comprises groups protecting its amino group at the alpha position (and if necessary, protection on its functional side groups).
At the end of this step, the group protecting the amino-terminal group is removed and the second amino acid, it too comprising the necessary protection, is bound.
The N-terminal protecting groups are removed after each amino acid has been bound; on the other hand, the protection is of course maintained on the side chains. When the polypeptide chain is complete, the peptide is cleaved from its support and the side protecting groups are removed.
The solid phase synthesis technique is well known to a person skilled in the art. See in particular Stewart et al. (1984) and Bodansky (1984).
The polypeptides obtained by chemical synthesis and which may comprise corresponding non-natural amino acids are also included in the invention.
The method for the production of a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultured under conditions allowing the expression of a recombinant polypeptide encoded by a nucleic acid sequence according to the invention, and in that the said recombinant polypeptide is recovered.
Also forming part of the invention is a method for the production of a heterologous polypeptide, characterized in that it uses a vector or a host cell containing at least one of the promoter and/or regulatory sequences according to the invention, or at least one of the sequences for cellular addressing according to the invention, or one of their fragments.
The recombinant polypeptides, characterized in that they are capable of being obtained by the said method of production, also form part of the invention.
The recombinant polypeptides obtained as indicated above may be both in glycosylated and nonglycosylated form and may or may not have the natural tertiary structure.
These polypeptides may be produced from the nucleic acid sequences defined above, according to techniques for the production of recombinant polypeptides known to persons skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.
An effective system of production of a recombinant polypeptide requires having a vector and a host cell according to the invention.
These cells may be obtained by introducing into the host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence.
The methods for the purification of a recombinant polypeptide which are used are known to persons skilled in the art. The recombinant polypeptide may be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatographic methods, immunoaffinity techniques with the aid of specific mono- or polyclonal antibodies, and the like.
A preferred variant consists in producing a recombinant polypeptide fused with a xe2x80x9ccarrierxe2x80x9d protein (chimeric protein). The advantage of this system is that it allows a stabilization and a reduction in proteolysis of the recombinant product, an increase in solubility during in vitro renaturation and/or simplification of the purification when the fusion partner has affinity for a specific ligand.
The mono- or polyclonal antibodies or fragments thereof, chimeric or immuno-conjugated antibodies, characterized in that they are capable of specifically recognizing a polypeptide or receptor according to the invention, also form part of the invention.
Specific polyclonal antibodies may be obtained from a serum of an animal immunized against, for example:
the LSR receptor purified from membranes of cells carrying the said LSR receptor, by methods well known to persons skilled in the art such as affinity chromatography using, for example, recombinant leptin as specific ligand, or
a polypeptide according to the invention, in particular produced by genetic recombination or by peptide synthesis, according to the customary procedures, from a nucleic acid sequence according to the invention.
There may be noted in particular the advantage of antibodies specifically recognizing certain polypeptides, variants or fragments, which are in particular biologically active, according to the invention.
The specific monoclonal antibodies may be obtained according to the conventional hybridoma culture method described by Kohler and Milstein, 1975.
The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F(abxe2x80x2)2 fragments. They may also be in the form of immunoconjugates or of labelled antibodies so as to obtain a detectable and/or quantifiable signal.
The invention also relates to methods for the detection and/or purification of a polypeptide according to the invention, characterized in that they use an antibody according to the invention.
The invention comprises, in addition, purified polypeptides, characterized in that they are obtained by a method according to the invention.
Moreover, in addition to their use for the purification of polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, may also be used for the detection of these polypeptides in a biological sample.
They thus constitute a means for the immunocytochemical or immunohistochemical analysis of the expression of the polypeptide of the LSR receptor on specific tissue sections, for example by immunofluorescence, gold labelling, enzymatic immunoconjugates.
They make it possible in particular to detect abnormal expression of these polypeptides in the biological tissues or samples, which makes them useful for the detection of abnormal expression of the LSR receptor or for monitoring the progress of the method of prevention or treatment.
More generally, the antibodies of the invention may be advantageously used in any situation where the expression of a polypeptide of the LSR receptor, normal or mutated, needs to be observed.
Also forming part of the invention are the methods for the determination of an allelic variability, a mutation, a deletion, a loss of heterozygosity or a genetic abnormality, characterized in that they use a nucleic acid sequence or an antibody according to the invention.
These methods relate to, for example, the methods for the diagnosis of predisposition to obesity, to the associated risks, or to pathologies associated with abnormalities in the metabolism of cytokines, by determining, in a biological sample from the patient, the presence of mutations in at least one of the sequences described above. The nucleic acid sequences analysed may be either the genomic DNA, the cDNA or the mRNA.
It will also be possible to use nucleic acids or antibodies based on the present invention in order to allow a positive and differential diagnosis in a patient taken in isolation. The nucleic sequences will be preferably used for a pre-symptomatic diagnosis in an at risk subject, in particular with a familial history. It is also possible to envisage an ante-natal diagnosis.
In addition, the detection of a specific mutation may allow an evolutive diagnosis, in particular as regards the intensity of the pathology or the probable period of its appearance.
The methods allowing the detection of a mutation in a gene compared with the natural gene are, of course, highly numerous. They can essentially be divided into two large categories. The first type of method is that in which the presence of a mutation is detected by comparing the mutated sequence with the corresponding nonmutated natural sequence, and the second type is that in which the presence of the mutation is detected indirectly, for example by evidence of the mismatches due to the presence of the mutation.
These methods can use the probes and primers of the present invention which are described. They are generally purified nucleic sequences for hybridization comprising at least 8 nucleotides, characterized in that they can hybridize specifically with a nucleic sequence chosen from the group comprising SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, SEQ ID 11, SEQ ID 13, SEQ ID 14 SEQ ID 15, SEQ ID 19 and SEQ ID 41. Preferably, the specific hybridization conditions are like those defined in the examples, or such that they ensure at least 95% homology. The length of these nucleic sequences for hybridization can vary from 8, 10, 15, 20 or 30 to 200 nucleotides, particularly from 20 to 50 nucleotides, more particularly from 20 to 30 nucleotides.
Among the methods for the determination of an allelic variability, a mutation, a deletion, a loss of heterozygocity or a genetic abnormality, the methods comprising at least one stage for the so-called PCR (polymerase chain reaction) or PCR-like amplification of the target sequence according to the invention likely to exhibit an abnormality with the aid of a pair of primers of nucleotide sequences according to the invention are preferred. The amplified products may be treated with the aid of an appropriate restriction enzyme before carrying out the detection or assay of the targeted product.
PCR-like will be understood to mean all methods using direct or indirect reproductions of nucleic acid sequences, or alternatively in which the labelling systems have been amplified, these techniques are of course known, in general they involve the amplification of DNA by a polymerase; when the original sample is an RNA, it is advisable to carry out a reverse transcription beforehand. There are currently a great number of methods allowing this amplification, for example the so-called NASBA xe2x80x9cNucleic Acid Sequence Based Amplificationxe2x80x9d (Compton 1991), TAS xe2x80x9cTranscription based Amplification Systemxe2x80x9d (Guatelli et al., 1990), LCR xe2x80x9cLigase Chain Reactionxe2x80x9d (Landegren et al., 1988), xe2x80x9cEndo Run Amplificationxe2x80x9d (ERA), xe2x80x9cCycling Probe Reactionxe2x80x9d (CPR), and SDA xe2x80x9cStrand Displacement Amplificationxe2x80x9d (Walker et al., 1992), methods well known to persons skilled in the art.
The invention comprises, in addition, methods for the diagnosis of pathologies and/or pathogeneses correlated with abnormal expression of a polypeptide and/or a receptor according to the invention, characterized in that an antibody according to the invention is brought into contact with the biological material to be tested, under conditions allowing the possible formation of specific immunological complexes between the said polypeptide and the said antibody, and in that the immunological complexes possibly formed are detected.
Mutations in one or more genes of the LSR complex may be responsible for various modifications of their product(s), which modifications can be used for a diagnostic approach. Indeed, modifications of antigenicity can allow the development of specific antibodies. The discrimination between the various conformations of LSR can be achieved by these methods. All these modifications may be used in a diagnostic approach by virtue of several well-known methods based on the use of mono- or polyclonal antibodies recognizing the normal polypeptide or mutated variants, such as for example using RIA or ELISA.
These diagnostic methods also relate to the methods of diagnosis by imaging in vivo or ex vivo using the monoclonal or polyclonal antibodies according to the invention, particularly those labelled and corresponding to all or part of the mutated polypeptides (imaging with the aid of antibodies coupled to a molecule which is detectable in PET-scan type imaging, for example).
Also included in the invention are the methods for selecting the chemical or biochemical compound capable of interacting, directly or indirectly, with the receptor according to the invention, and/or allowing the expression or the activity of the said receptor to be modulated, characterized in that they use a receptor, a nucleic acid, a polypeptide, a vector, a cell or a mammal according to the invention.
The invention relates to a method for screening compounds modifying the activity of the LSR receptor, consisting in measuring the effect of candidate compounds on various parameters reflecting, directly or indirectly, taken independently or in combination, an LSR receptor activity.
For the screening of compounds capable of modulating the LSR activity for lipoprotein clearance, the preferred principal effect is the effect of the compound on the activity of binding, internalization and degradation of the lipoproteins by the LSR receptor.
This effect can be analysed in the absence or in the presence of free fatty acids, or of any other agent known to induce or to inhibit the activity of LSR on the clearance of lipoproteins, or in the absence or the presence of leptin, or of any other agent capable of inducing or of inhibiting the LSR function of cytokine clearance. It can, in addition, be measured in the absence or in the presence of agents capable of promoting or reducing the lipase activities, either intracellular or extracellular, as well as in the presence or in the absence of alternative known routes of degradation of lipoproteins.
Various indirect parameters can also be measured, including the following
the change in weight induced by the administration of the compound
the food intake induced by the administration of the compound
the postprandial lipemic response induced by the administration of the compound, before, during or after ingestion of a meal, for example high in fat.
The selection of compounds capable of influencing the plasma triglyceride concentrations, and/or the binding, internalization and hepatic degradation of lipoproteins or particles high in triglycerides, will be preferred.
For the screening of compounds capable of modulating the LSR activity of clearance of cytokines, in particular of leptin, the preferred principal effect is the effect of the compound on the activity of binding, internalization and hepatic degradation of cytokines by the LSR receptor, in the absence or in the presence of free fatty acids.
The measurement of the binding, internalization and/or degradation of lipids or of cytokines can be carried out, for example, on hepatocytes or fibroblasts in culture, or on any other cell expressing the LSR receptor at its surface. The cells will be preferably cells expressing a recombinant LSR receptor, more particularly cells expressing a recombinant LSR receptor and whose endogenous LSR receptor would be inactivated or absent. These cells may or may not express the LDL receptor.
The screening of compounds modulating the LSR activity preferably uses cells or model animals according to the invention, in particular mice, rats or humans, more particularly those described above and in the examples which follow.
Screening may be used to test compounds capable of modifying the level and/or the specificity of expression of the LSR receptor either by binding competitively to the sites for binding of trariscription factors situated in the LSR-promoter or by binding directly to the transcription factors.
The level of expression of the LSR receptor and its location can be analysed by hybridization in solution with large probes as indicated in Patent PCT WO 97/05277, the teaching of this document being incorporated by reference. Briefly, a cDNA or the genomic DNA for the LSR receptor or alternatively a fragment thereof is inserted at a cloning site situated directly downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter in order to produce an antisense RNA. Preferably, the insert comprises at least 100 consecutive nucleotides of the genomic sequence of the LSR receptor or of one of the cDNAs of the present invention, more particularly one or more of the cDNAs of SEQ ID 9, SEQ ID 11 or SEQ ID 13. The plasmid is linearized and transcribed in the presence of ribonucleotides comprising modified ribonucleotides such as Biotin-UTP and Digoxigenin-UTP. An excess of this labelled RNA is hybridized in solution with the mRNAs isolated from cells or from tissues of interest. The hybridizations are carried out under stringent conditions (40-50xc2x0 C. for 16 h in a solution containing 80% formamide and 0.4 M NaCl, pH 7-8). The non-hybridized probe is eliminated by digestion with ribonucleases specific for single-stranded RNAs (CL3, T1, PhyM, U2 or A RNases). The presence of modified nucleotides biotin-UTP allows the capture of the hybrids on microtitre plates carrying streptavidine. The presence of the DIG modification allows the detection and quantification of the hybrids by ELISA using anti-DIG antibodies coupled to alkaline phosphatase.
A quantitative analysis of the expression of the gene for the LSR receptor can also be carried out using DNA templates, the term DNA templates designating a one-dimensional, two-dimensional or multi-dimensional arrangement of a plurality of nucleic acids having a sufficient length to allow a specific detection of the expression of mRNAs capable of hybridizing thereto. For example, the DNA templates may contain a plurality of nucleic acids derived from genes for which it is desired to estimate the level of expression. The DNA templates may include the genomic sequences of LSR, that of a cDNA of the present invention, more particuliarly one or more of the cDNAs of SEQ ID 9, SEQ ID 11 or SEQ ID 13, any sequences complementary thereto or any fragments thereof. Preferably, the fragments comprise at least 15, at least 25, at least 50, at least 100 or at least 500 consecutive nucleotides of the nucleic sequences from which they are derived.
For example, a quantitative analysis of the expression of the LSR receptor can be carried out with a DNA template having the cDNA for the LSR receptor as described in Schena et al. (1995 and 1996). cDNAs for the LSR receptor or fragments thereof are amplified by PCR and bound in the form of a template from a 96-well microplate onto a sylated microscope slide using a very fast automated machine. The DNA template thus produced is incubated in a humid chamber in order to allow its rehydratation. It is then rinsed once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in a sodium borohydride solution. The template is then submerged in water for 2 min at 95xc2x0 C., transferred into 0.2% SDS for 1 min, rinsed twice with water, dried and stored in the dark at 25xc2x0 C.
The mRNAs of cells and of tissues are isolated or obtained from a commercial source, for example the company Clontech. The probes are prepared by a reverse transcription cycle. The probes are then hybridized with the DNA template of 1 cm2 under a glass coverslip of 14xc3x9714 mm for 6-12 hours at 60xc2x0 C. The template is washed for 5 min at 25xc2x0 C. in a washing buffer at low stringency (1xc3x97SSC/0.2% SDS) and then for 10 min at room temperature in a highly stringent buffer (0.1xc3x97SSC/0.2% SDS). The template is analysed in 0.1xc3x97SSC using a laser fluorescence microscope with a set of appropriate filters. Measurements of precise differential expression are obtained by taking the mean of the ratios of two independent hybridizations.
A quantitative analysis of the expression of the LSR receptor can also be carried out with cDNAs for the LSR receptor or fragments thereof on DNA templates according to the description by Pietu et al. (1996). The cDNAs for the LSR receptor or fragments thereof are amplified by PCR and bound to membranes. The mRNAs obtained from different tissues or cells are labelled with radioactive nucleotides. After hybridization and washing under controlled conditions, the hybridized mRNAs are detected with a Phosphor Imager or by autoradiography. The experiments are carried out in duplicate and a quantitative analysis of the differentially expressed mRNAs can be carried out.
Alternatively, the analysis of the expression of the LSR receptor can be made with DNA templates at high density as described by Lockhart et al. (1996) and Sosnowski et al. (1997). Oligonucleotides of 15 to 50 nucleotides, preferably about 20 nucleotides, extracted from genomic DNA or cDNA sequences for the LSR receptor or of their complementary sequences are synthesized directly on a chip or synthesized and then addressed onto the chip.
LSR cDNA probes labelled with an appropriate compound such as biotin, digoxigenin or a fluorescent molecule are synthesized from a population of mRNA and are fragmented into oligonucleotides of 50 to 100 nucleotides on average. The probes thus obtained are then hybridized to a chip. After washing as described in Lockhart et al (1996) and an application of various electric fields (Sosnowski et al. 1997), the labelled compounds are detected and quantitied. The hybridizations are duplicated. A comparative analysis of the intensity of the signals generated by the probes on the same target oligonucleotide in various cDNA samples indicates a differential expression of the mRNAs for the LSR receptor.
The techniques mentioned above allow the analysis of the levels of expression of the LSR receptor, in the same cell or the same tissue depending on various conditions, for example of induction or of noninduction, but also the analysis of the tissue specificity of this expression, under conditions which can also vary. It will be possible, by virtue of these techniques, to analyse the expression of either of the subunits of the LSR receptor, and more generally of different forms derived from alternative splicing, by adequately defining the probes.
The effect of compounds which are candidates for modulating the level or the specificity of expression, or of splicing of the different forms of the LSR receptor can thus be analysed on a large scale by exposing the cells which are the source of messenger RNA, in particular the model cells according to the invention, whether they express LSR naturally or whether they are recombinant cells, to the said candidate compounds.
Another aspect of the present invention consists in methods of identifying molecules capable of binding to the LSR receptor. Such molecules can be used to modulate the activity of the LSR receptor. For example, such molecules can be used to stimulate or reduce the degradation of lipoproteins, preferably of lipoproteins high in triglycerides, or of cytokines, preferably of leptin. Such molecules can also be used to inhibit the activation by leptin or the activation by free fatty acids of the LSR activity.
Numerous methods exist for identifying ligands for the LSR receptor. One of these methods is described in U.S. Pat. No. 5,270,170, whose teaching is incorporated by reference. Briefly, a library is constructed which consists of random peptides, comprising a plurality of vectors each encoding a fusion between a peptide which is a candidate for binding to the LSR receptor and a protein binding to DNA such as the Lac repressor encoded by the lad gene. The vectors for the library of random peptides also contain binding sites for the proteins binding to DNA such as the LacO site when the protein is the Lac repressor. The library of random peptides is introduced into a host cell in which the fusion protein is expressed. The host cell is then lysed under conditions allowing the binding of the fusion protein to the sites of the vector.
The vectors which have bound the fusion protein are brought into contact with the immobilized LSR receptor, a subunit of the immobilized LSR receptor or a fragment of the immobilized LSR receptor under conditions allowing the peptides to bind specifically. For example, the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids can be immobilized by binding to a surface such as a plate or a plastic particle.
The vectors which encode the peptides capable of binding to the LSR receptor are specifically retained at the surface by interactions between the peptide and the LSR receptor, a subunit of the receptor or a fragment thereof.
Alternatively, molecules capable of interacting with the LSR receptor can be identified using a double hybrid system such as the Matchmaker Two Hybrid System 2. According to the instructions of the manual accompanying the Matchmaker Two Hybrid System 2 (Catalogue No. K1604-1, Clontech), whose teaching is incorporated by reference, the nucleic acids encoding the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids are inserted into an expression vector so that they are in phase with the DNA encoding the DNA binding domain of the transcription activator of yeast GAL4. The nucleic acids of a library encoding proteins or peptides capable of interacting with the LSR receptor are inserted into a second expression vector so that they are in phase with the DNA encoding the activation domain of the GAL4 activator. The yeasts are transformed with the two expression plasmids and they are placed in a medium which makes it possible to select the cells expressing markers contained in each of the vectors as well as those expressing the HIS3 gene whose expression is dependent on GAL4. The transformed cells capable of growing on a histidine-free medium are analysed for expression of LacZ under the dependence of GAL4. The cells which grow in the absence of histidine and express LacZ contain a plasmid which encodes proteins or peptides which interact with the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids thereof.
To study the interaction of the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids with small molecules such as those generated by combinatory chemistry, it is possible to use an HPLC-coupled microdialysis as described in Wang et al. (1997), or an affinity capillary electrophoresis as described in Busch et al. (1997), the teaching of these documents being incorporated by reference.
In other methods, the peptides or small molecules capable of interacting with the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids may be linked to detectable markers such as radioactive, fluorescent or enzymatic markers. These labelled molecules are brought into contact with the immobilized LSR receptor, an immobilized subunit thereof or an immobilized fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids under conditions allowing a specific interaction. After elimination of the molecules which are not specifically bound, the bound molecules are detected by appropriate means.
These methods may allow in particular the identification of fatty acids or analogues capable of binding to the fatty acid binding site on the LSR, of lipoproteins or analogues, capable of binding to the lipoprotein binding site on the LSR receptor, of leptin derivatives or analogues capable of binding to the leptin binding site on the LSR, and of derivatives of the gC1qR receptor or analogues capable of binding to the gC1qR binding site on the LSR.
In addition, the peptides or small molecules which bind to LSR, preferably to the binding sites on the LSR receptor for fatty acids, lipoproteins, cytokines, in particular leptin, or gC1qR or one of its analogous proteins, can be identified by competition experiments. In such experiments, the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids is immobilized on a surface such as a plastic support. Increasing quantities of peptides or of small molecules are brought into contact with the immobilized LSR receptor, an immobilized subunit thereof or an immobilized fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids in the presence of a labelled ligand for the receptor, it being possible for this ligand to be, for example, leptin, oleate, the LDLs or gC1qR. The ligand for the LSR receptor may be labelled with a radioactive, fluorescent or enzymatic marker. The capacity of the molecule tested to interact with the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids is determined by measuring the quantity of labelled ligand bound in the presence of the molecule tested. A decrease in the quantity of bound ligand when the molecule tested is present indicates that the latter is capable of interacting with the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids.
These methods can in particular allow the identification of fatty acids or analogues capable of binding to the fatty acid binding site on the LSR, of lipoproteins or analogues, capable of binding to the lipoprotein binding site on the LSR receptor, of leptin derivatives or analogues capable of binding to the leptin binding site on the LSR, and of derivatives of the gC1qR receptor or analogues capable of binding to the gC1qR binding site on the LSR. The capacity of such compounds, or of any other candidate compound, to compete with the binding of oleates, lipoproteins, leptin or gC1qR to LSR can be measured in particular.
The BIACORE technology can also be used to carry out the screening of compounds capable of interacting with the LSR receptor. This technology is described in Szabo et al. (1995) and in Edwards and Leartherbarrow (1997), of which the teaching is incorporated by reference, and makes it possible to detect interactions between molecules in real time without the use of labelling. It is based on the phenomenon of SPR (surface plasmon resonance). Briefly, the molecule to be analysed is bound to a surface (typically using a carboxymethyl dextran matrix). A light ray is directed onto the face of the surface which does not contain the sample and is reflected by the said surface. The SPR phenomenon causes a reduction in the intensity of the reflected light with a specific combination of angle and of wavelength. The molecule binding events cause a change in the refractive index at the surface which is detected as a modification of the SPR signal. To carry out a screening of compounds capable of interacting with the LSR receptor, the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids, is immobilized on a surface. This surface constitutes one face of a cell through which passes the molecule to be tested. The binding of the molecule to the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids is detected by a change in the SPR signal. The molecules tested may be proteins, peptides, carbohydrates, lipids or small molecules generated, for example, by combinatory chemistry. The candidate proteins can be extracted from any tissue, obtained from any species. The BIACORE technology can also be used by immobilizing eukaryotic or prokaryotic cells or lipid vesicles having an endogenous or recombinant LSR receptor at their surface.
One of the main advantages of this method is that it allows the determination of the association constants between the LSR receptor and the interacting molecules. Thus, it is possible to specifically select the molecules interacting with high or low association constants.
The proteins or other molecules interacting with the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than consecutive amino acids can be identified using affinity columns which contain the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids. The LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids may be attached to the column using conventional techniques including chemical coupling to an appropriate column matrix such as agarose, Affi Gel, or other matrices known to a person skilled in the art. In another aspect of the invention, the affinity column may contain chimeric proteins in which the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids would be fused, for example, with glutathione S-transferase. The molecules to be tested which are described above are then deposited on the column. The molecules interacting with the LSR receptor, a subunit thereof or a fragment thereof comprising at least 10, at least 20, at least 30, or more than 30 consecutive amino acids are retained by the column and can be isolated by elution. In the case where the molecules tested are proteins, they can then be analysed on a 2-D electrophoresis gel as described in Ramunsen et al. (1997), of which the teaching is incorporated by reference. Alternatively, the proteins or the other molecules retained by the affinity column can be purified by electrophoresis and sequenced. A similar method can be used to isolate antibodies, to screen  less than  less than phage display greater than  greater than  products or  less than  less than phage display greater than  greater than  derived human antibodies.
The invention also relates to a method of screening compounds interacting with the promoter and/or regulatory sequences of the LSR receptor.
The nucleic acids encoding proteins interacting with the promoter and/or regulatory sequences of the LSR receptor gene, more particularly a nucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, can be identified using a single hybrid system such as that described in the manual accompanying the Matchmaker One-Hybrid System from Clontech (Catalogue No. K1603-1), of which the teaching is incorporated by reference. Briefly, the target nucleotide sequence is cloned upstream of a selectable marker gene and integrated into a yeast genome. The yeasts containing the integrated marker gene are transformed by a library containing fusions between cDNAs encoding candidate proteins for binding to the promoter and/or regulatory regions of the gene for the LSR receptor and the yeast transcription factor activating domain such as GAL4. The yeasts are placed in a medium which makes it possible to select the cells expressing the marker gene. The yeasts selected contain a fusion protein capable of binding to the promoter and/or regulatory target region. The cDNAs of the genes encoding the fusion proteins are then sequenced. The corresponding inserts can then be cloned into expression or transcription vectors in vitro. The binding of the polypeptides thus encoded to the promoter target sequences can be confirmed by techniques familiar to persons skilled in the art, including gel retardation or protection to DNAse experiments.
The screening of compounds capable of modifying the expression of the LSR receptor by binding to its regulatory and/or promoter sequences can also be carried out with the aid of  less than  less than reporter greater than  greater than  genes. For example, a genomic region situated in 5xe2x80x2 of the coding sequence of the LSR receptor, more particularly a nucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, can be cloned into a vector such as pSEAP-Basic, pSEAP-Enhancer, pxcex2gal-Basic, pxcex2gal-Enhancer, or pEGFP-1 available from Clontech. Briefly, each of these vectors contains multiple cloning sites situated upstream of a marker gene encoding an easily detectable protein such as alkaline phosphatase, xcex2-galactosidase or GFP (green fluorescent protein). After insertion of the genomic region situated in 5xe2x80x2 of the coding sequence of the LSR receptor, more particularly a nucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, the level of expression of the marker proteins is measured and compared with a vector containing no insert. The effect of candidate compounds on the expression resulting from the regulatory and/or promoter sequences of LSR can thus be evaluated.
The screening of the compounds capable of binding to the regulatory and/or promoter regions of the gene for the LSR receptor can also be carried out by gel retardation experiments well known to persons skilled in the art and described in Fried and Crothers (1981), Garner and Revzin (1981) and Dent and Latchman (1993), of which the teaching is incorporated by reference. These experiments are based on the principle that a DNA fragment bound to a protein migrates more slowly than the same fragment without protein. Briefly, the target nucleotide sequence is labelled. It is then brought into contact either with a nuclear or total cell extract prepared so as to contain the transcription factors, or with various compounds to be tested. The interaction between the regulatory and/or promoter region of the gene for the LSR receptor and the transcription factor or compound is detected after electrophoresis by retardation of migration.
The chemical or biochemical compounds, characterized in that they make it possible to modulate the expression or the activity of the receptor according to the invention, also form part of the invention.
The chemical or biochemical compounds, characterized in that they are capable of interacting, directly or indirectly, with the receptor according to the invention, also form part of the invention.
The chemical or biochemical compounds, characterized in that they are selected by the said methods defined above, also form part of the invention.
In particular, among these compounds according to the invention, a leptin or one of its derived compounds, preferably one of its protein variants, or leptins which are chemically modified or which are obtained by genetic recombination, or one of their fragments, are preferred.
Compounds which make it possible to modulate the expression or the activity of the receptor are understood to mean the compounds which make it possible in particular to reduce, stabilize or increase the number, the recycling rate and/or the change in the conformation of the receptor according to the invention, or to promote or inhibit the overall activity or the activity of one of the domains of the said receptor or alternatively to reestablish normal expression of the said receptor in the case, for example, where a genetic abnormality is observed. These compounds may, for example, interact as ligands specific for the said receptor or for one of its domains as cofactor, or as inhibitor, in particular a competitive inhibitor, or alternatively having an agonist or antagonist activity on the conformational changes in the complex. These compounds may also interact by neutralizing the natural ligands specific for the said receptor and by thereby inhibiting the receptor activity induced by these ligands.
Among these compounds, the compounds which make it possible to modulate the number of polypeptides of the said receptor, its recycling rate and/or the selectivity of their activity, are preferred.
Also preferred are the compounds according to the invention, characterized in that they allow an increase in the total activity or in the expression of the receptor according to the invention, and/or a specific increase in the clearance activity for cytokines, in particular leptin, of the said receptor, and/or a specific increase in the clearance activity for lipoproteins, of the said receptor.
Also preferred are the compounds characterized in that they allow a decrease in the total activity or in the expression of the receptor according to the invention, and/or a specific decrease in the clearance activity for cytokines, in particular leptin, of the said receptor, and/or a specific decrease in the clearance activity for lipoproteins, of the said receptor.
Also preferred are the compounds characterized in that they allow modulation of the elimination of the cytokines, in particular leptin, and/or modulation of the elimination of the lipoproteins, chylomicron residues, and/or triglycerides.
The invention also comprises the compounds according to the invention, characterized in that they allow modulation of the level of cytokines, in particular leptinemia, and/or modulation of the level of lipoproteins, chylomicron residues, and/or triglycerides.
The compounds according to the invention, characterized in that they allow control of the level of cytokines, in particular leptinemia, are more particularly preferred.
Still preferably, the invention comprises the compounds according to the invention, characterized in that they allow control, preferably a decrease, of the level of lipoproteins, a decrease in the plasma concentration of chylomicron residues, and/or a decrease in triglyceridemia.
Among the compounds which are most preferred, there are preferred those characterized in that they are chosen from:
a. an antibody according to the invention;
b. a polypeptide according to the invention;
c. a polypeptide according to the invention, characterized in that it corresponds to a soluble form of the receptor according to the invention;
d. a vector according to the invention;
e. a vector according to the invention, characterized in that it has on its outer surface a site for specific recognition of hepatic cells;
f. a vector according to the invention, characterized in that the product of expression of the nucleic acid inserted by the vector into the target cell is either anchored in or excreted by the said transformed target cell;
g. a sense or antisense oligonucleotide according to the invention;
h. a leptin, or one of its protein variants, or a leptin which is chemically modified or which is modified by genetic recombination, or one of their fragments.
The invention finally relates to the compounds according to the invention as a medicament.
The compounds according to the invention as a medicament for the prevention and/or treatment of pathologies and/or of pathogeneses linked to disorders in dietary habit are preferred in particular.
The compounds according to the invention as a medicament for the prevention and/or treatment of pathologies and/or of pathogeneses linked to disorders in the metabolism of cytokines are also preferred.
Preferably, the invention also relates to the compounds according to the invention as medicament for the prevention or treatment of obesity or anorexia.
The compounds according to the invention as a medicament for the prevention and/or treatment of pathologies and/or of pathogeneses associated with, or induced by obesity, are the preferred compounds.
In particular, there are preferred the compounds according to the invention, as a medicament for the prevention and/or treatment of cardiac insufficiency, of coronary insufficiency, of cerebrovascular accidents, of atheromatous disease, of atherosclerosis, of high blood pressure, of non-insulin-dependent diabetes, of hyperlipidemia and/or of hyperuricemia.
The most preferred are the compounds according to the invention, as a medicament for the prevention and/or treatment of atheromatous disease and/or of atherosclerosis.
Finally, the invention comprises compounds according to the invention for the prevention and/or treatment by gene therapy, of pathologies and/or of pathogeneses linked to disorders in dietary habit, of obesity and/or of pathologies and/or of pathogeneses associated with, or induced by, obesity.
The compounds of the invention as active ingredients of a medicament will be preferably in soluble form, combined with a pharmaceutically acceptable vehicle.
Such compounds which can be used as a medicament offer a new approach for preventing and/or treating pathologies and/or pathogeneses linked to disorders in dietary habit such as obesity or anorexia, and the related risks and/or complications.
Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular or intradermal route or by the oral route.
Their modes of administration, optimum dosages and galenic forms can be determined according to the criteria generally taken into account in establishing a treatment suited to a patient, such as for example the age or body weight of the patient, the seriousness of his general condition, the tolerance to treatment and the side effects observed, and the like.
As mentioned above, depending on the cases, it may be advisable to amplify the activity of LSR, by promoting, for example, the expression of its genes or by increasing the activity of their expression products, in pathological cases resulting from the fact that at least one of these genes is not expressed, is insufficiently expressed or is expressed in an abnormal form which does not allow the expression product to carry out its functions, or on the contrary to repress an overexpression or an abnormal expression of these genes. It is therefore advisable in general to compensate for the deficiency or the overexpression of expression products of this gene by a so-called xe2x80x9creplacementxe2x80x9d therapy allowing the amplification or the reduction in the activities of the LSR complex.
The replacement therapy may be carried out by gene therapy, that is to say by introducing the nucleic acid sequences according to the invention and/or the corresponding genes with the elements which allow their expression in vivo, in the case where one of the genes is insufficiently expressed for example, or alternatively when it is expressed in an abnormal form.
The principles of gene therapy are known. It is possible to use viral vectors according to the invention; it is also possible to envisage nonviral, that is to say synthetic, vectors which mimic viral sequences or alternatively which consist of naked RNA or DNA according to the technique developed in particular by the company VICAL.
In most cases, it is necessary to envisage targeting elements ensuring expression specific for the liver so as to be able to limit the zones of expression of the proteins which remain involved in the clearance of leptin and that of lipoproteins. It is even advantageous, in some cases, to have vectors for transient expression or at least for controlled expression which it will be possible to block when necessary.